Autologous chimeric antigen receptor (CAR) T cell therapies have shown significant success in treating hematological malignancies. However, challenges such as high costs, lengthy vein-to-vein times, and manufacturing uncertainties due to variations in autologous T cell characteristics continue to hinder widespread access to CAR T therapy.

Exploring Off-the-Shelf Allogeneic CAR T Cells

To address these issues, researchers are exploring the use of off-the-shelf allogeneic CAR T cells derived from healthy donors. Allogeneic CAR T cells offer many potential advantages, including reduced costs, quicker patient treatment, predefined product characteristics, and secured cell doses. However, they also present challenges related to patient safety associated with allogeneic cell therapy, besides the challenge of process scalability and establishing automated workflows. The major safety issues are related to the risks of graft-versus-host disease (GVHD) and CAR T cell graft rejection by the host immune system, stemming from the alloreactivity of donor T cells. This blog explores strategies to develop allogeneic CAR T cells with reduced GVHD risk and extended persistence in patients, as well as the platforms and tools at OmniaBio that facilitate these strategies in the manufacturing process.

Cell Source Considerations to Reduce Cell Product Alloreactivity 

Currently, T cells used for autologous CAR T cell manufacturing are mainly derived from peripheral blood mononuclear cells (PBMCs). The T cell receptor (TCR) in donor alpha beta (αβ) T cells recognizes peptides presented by major histocompatibility complex (MHC) of host cells is essential to the pathogenesis of GVHD. Additionally, the host immune system's recognition of donor MHC class I molecules mediates allogeneic CAR T cell rejection. A crucial factor in allogeneic CAR T cell manufacturing is minimizing the alloreactivity of the final cell product. Alternative cell sources being investigated include:

1. T cells from MHC-matched donors;
2. Unconventional sources like umbilical cord blood (UCB) or induced pluripotent stem cells (iPSCs);
3. Non-αβ T cells such as gamma delta (γδ) T cells, or CD4 and CD8 double negative T cells

Clinical trial data using donor-derived allogeneic CAR T cells for the treatment of relapsed/refractory B-cell precursor acute lymphoblastic leukemia have shown promising safety and efficacy. Preclinical results from allogeneic CAR T cells manufactured from non-αβ T cells also indicate reduced GVHD risk and scalability (1,2,3).

Gene Editing Approaches to Reduce GVHD and Extend the Persistence of Allogeneic CAR T Cells

Gene editing technologies are paving the way for the next generation of adoptive cellular therapies. Strategies being explored to eliminate GVHD and make allogeneic CAR T cells less recognizable to the host immune system include:

1. Targeted integration approaches to incorporate the CAR construct into the T cell receptor constant α chain (TRAC) locus, simultaneously abolishing TCR expression and integrating CAR. 
2. Deletion of the beta-2 (β2) microglobulin gene, which is essential for forming functional MHC class I molecules on the cell surface, to overcome host T cell-mediated rejection of allogeneic cell products. 
3. Deleting CD52 expression to enhance resistance to lymphodepletion treatment, where anti-CD52 monoclonal antibodies are used to prevent allo-rejection by eliminating host T cells expressing CD52; 

Additionally, other strategies are being explored to prolong the persistence of allogeneic CAR-T cells in vivo, including:

4. Chimeric Receptor Expression: Creating diverse chimeric receptors that convert inhibitory signals from the tumor microenvironment into activating signals. For example, receptors that combine the truncated extracellular domain of PD1 with the transmembrane and cytoplasmic signaling domains of CD28 transform immune-suppressive signals into activating signals, thereby enhancing CAR T cell activity. 
5. Secretory Cytokine Expression: Engineering CAR T cells to secrete cytokines that improve their survival. 

Preliminary clinical trial results using gene-edited allogeneic CAR T cells, such as those generated by CRISPR–Cas9, TALEN, or megaTAL nucleases, are encouraging. They demonstrate a manageable safety profile and durable complete responses in relapsed B-cell acute lymphoblastic leukemia, relapsed and refractory myeloma, CD7-positive hematological malignancies, and some solid tumours, supporting the feasibility and safety of allogeneic CAR T cell therapy in these circumstances (5,6,7,8).

Addressing Manufacturing Challenges

While the development of off-the-shelf CAR T cell-based therapies holds transformative potential, manufacturing challenges remain in establishing suitable automated and scalable workflows using alternative cell sources and safety gene editing approaches. Understanding application, product and process requirements, alongside access to a plug-and-play toolbox, can help select a sustainable manufacturing strategy.

• Gene Editing Technologies: Gene editing is a crucial part of the manufacturing workflow to enable these allogeneic cell therapies to be safe and efficacious. There are numerous gene editing solutions including transposases, Talens, zinc fingers and several genome editing technologies such as CRISPR-Cas9, but they all come with unknown risks and varying editing efficiencies as well as licensing issues when moving to GMP manufacturing. Having a clearly defined strategy, and an established path towards CMC in your development, is important in reducing time and costs.
• Utilizing Diverse Gene Delivery Methods: Innovative gene delivery methods are being explored to enhance the efficiency and safety of inserting CAR genes into T cells. They include lentiviral vectors, adenoviral vectors, lipid nanoparticles and electroporation. Viral vectors have been the most commonly used for gene delivery, but they come with risks and disadvantages such as insertional mutagenesis and high production costs. Recently, the U.S. FDA stated that all commercial CAR T cell therapies must have a box warning to indicate there is a risk of secondary cancers that can arise due to the insertional mutagenesis from the viral vectors used. Although this risk is low, there is increased interest in using non-viral gene delivery mechanisms such as electroporation and liquid nanoparticles (LNPs). No matter what delivery mechanism is used, scalable, cost-effective, and safe gene delivery mechanisms should be developed to enable the consistent production of high-quality CAR T cells.
• Incorporating Advanced Platforms for Manufacturing: To streamline the manufacturing process for allogeneic CAR T therapies, the industry is moving towards automated and closed systems. Advanced bioreactors equipped with real-time monitoring and control capabilities allow for precise regulation of cell culture conditions, ensuring consistent cell quality and yield. Integrating automated, closed systems for other steps within the allogeneic CAR T workflow is important as it reduces manual intervention and contamination risk. Implementing modular and flexible manufacturing platforms enables scalability and adaptability to different production scales.

In summary, while autologous CAR T cell therapies have paved the way for groundbreaking advances in cancer treatment, the pursuit of off-the-shelf allogeneic CAR T cells represents a promising evolution in the field. By addressing challenges related to cost, production efficiency, and patient safety, allogeneic CAR T cell therapies have the potential to make these therapies more accessible and consistent. Continued research and development, particularly in strategies to minimize GVHD, improve cell persistence, and address manufacturing challenges, and moving suitable approaches into the clinic, will be crucial for realizing the full potential of allogeneic CAR T cell therapies. The approaches and clinical data reviewed herein and the established manufacturing platforms at OmniaBio exemplify the strides being made to overcome these hurdles, setting the stage for a new era in cellular therapy that could significantly enhance patient outcomes and broaden treatment options. 

Does your company require help overcoming obstacles with your allogeneic CAR T cell therapy program? OmniaBio has industry-leading expertise supporting numerous therapeutic developers across the entire product lifecycle from process optimization through scale-up to GMP-compliant manufacturing. Learn more about how we can help your company here.

Reference

1. Francesca Del Bufalo, Marco Becilli, Chiara Rosignoli, etc. Allogeneic, donor-derived, second-generation, CD19-directed CAR-T cells for the treatment of pediatric relapsed/refractory BCP-ALL. Blood. 2023;142(2):146-157. PMID: 37172203. DOI: 10.1182/blood.2023020023
2. Daniel Vasic, Jong Bok Lee, Yuki Leung,etc. Allogeneic double-negative CAR-T cells inhibit tumor growth without off-tumor toxicities.Sci Immunol.2022;7(70). PMID: 35452255. DOI: 10.1126/sciimmunol.abl3642
3. Diego Sánchez Martínez, Néstor Tirado, Sofia Mensurado. Generation and proof-of-concept for allogeneic CD123 CAR-Delta One T (DOT) cells in acute myeloid leukemia. J Immunother Cancer. 2022;10(9):e005400. PMID: 36162920. PMCID: PMC9516293. DOI: 10.1136/jitc-2022-005400
4. Xiaojun Liu, Raghuveer Ranganathan, Shuguang Jiang. A Chimeric Switch-Receptor Targeting PD1 Augments the Efficacy of Second-Generation CAR T Cells in Advanced Solid Tumors. Cancer Res. 2016 Mar 15;76(6):1578-90. PMID: 26979791. PMCID: PMC4800826 DOI: 10.1158/0008-5472.CAN-15-2524
5. Sham Mailankody, Jeffrey V Matous, Saurabh Chhabra. Allogeneic BCMA-targeting CAR-T cells in relapsed/refractory multiple myeloma: phase 1 UNIVERSAL trial interim results. Clinical Trial Nat Med. 2023;29(2):422-429. PMID: 36690811. DOI: 10.1038/s41591-022-02182-7
6. Reuben Benjamin, Nitin Jain, Marcela V Maus, etc. UCART19, a first-in-class allogeneic anti-CD19 chimeric antigen receptor T-cell therapy for adults with relapsed or refractory B-cell acute lymphoblastic leukaemia (CALM): a phase 1, dose-escalation trial. Lancet Haematol, 2022 Nov;9(11):e833-e843. PMID: 36228643. DOI: 10.1016/S2352-3026(22)00245-9
7. Sumanta K Pal, Ben Tran, John B A G Haanen, etc. CD70-Targeted Allogeneic CAR-T-Cell Therapy for Advanced Clear Cell Renal Cell Carcinoma.  Cancer Discov. 2024 Jul 1;14(7):1176-1189. PMID: 3858318 PMCID: PMC11215406. DOI: 10.1158/2159-8290.CD-24-0102
8. Giorgio Ottaviano, Christos Georgiadis, Soragia Athina Gkazi, etc. Phase 1 clinical trial of CRISPR-engineered CAR19 universal T cells for treatment of children with refractory B cell leukemia. Sci Transl Med, 2022 Oct 26;14(668). PMID: 36288281. DOI: 10.1126/scitranslmed.abq3010

About The Authors

Yanling Liu, MD, PhD, Senior Scientist, Cytiva

Yanling Liu is a senior scientist at Cytiva. She obtained her PhD degree at Huazhong University of Science and Technology, and MD at Tongji Medical University, where she worked as a hematologist for several years. She started her career in Canada as a scientist and spent over a decade at the University of Toronto researching human memory B cell differentiation and developing therapeutic antibodies for blood malignancies. She then joined Cytiva in 2022 and, over the past two years, she has been involved in CAR T cell manufacturing and NK cell manufacturing projects, providing extensive expertise in developing the technology platform to commercialize through OmniaBio and to streamline therapeutic development clients.

Paul Bowles, PhD, Associate Principal Scientist, Cell and Immunotherapies, OmniaBio Inc. 

Since obtaining his PhD in immunology, Paul has spent more than a decade in cell and gene therapy process development. Paul started his industrial career working first at GE Healthcare and then as part of a collaboration between Cytiva and CCRM, where he worked on process development of various immunotherapy platforms. Paul developed several closed, semi-automated processes, a CAR T cell, and an antigen-specific T cell process that went into Phase I/II clinical trials. He then spent time working at Treadwell Therapeutics, leading the process and analytical team focused on TCR-T therapies. Paul currently works as an Associate Principal Scientist and co-lead of the Immuno Cell Therapy platform, responsible for developing robust platform processes that OmniaBio offers to benefit therapeutic sponsors.